Mammalian cell culture is the system of choice for many recombinant protein production processes due to its ability to produce proteins with proper post-translational modifications. With increasing manufacturing demand, a strong motivation exists to improve process efficiency by increasing product yield. Attaining the grams per liter production levels of biotherapeutics in commercial production processes relies upon the optimization of both mammalian cell culture and engineering methods.
Inherent in current high density, protein-free mammalian cell cultures is the problem of cell death of which apoptosis can account for up to 80% in a typical fed-batch bioreactor, induced in response to stressors such as nutrient and growth factor deprivation, oxygen depletion, toxin accumulation, and shear stress (Goswami et al., Biotechnol. Bioeng. 62:632-640 (1999)). Apoptosis limits the maximum viable cell density, accelerates the onset of the death phase and potentially decreases heterologous protein yield (Chiang and Sisk, Biotechnol. Bioeng. 91:779-792 (2005); Figueroa et al., Biotechnol. Bioeng. 73:211-222 (2001), Metab. Eng. 5:230-245 (2003), Biotechnol Bioeng. 85:589-600 (2004); Mercille and Massie, Biotechnol. Bioeng. 44:1140-1154 (1994)).
Apoptosis is a result of a complex network of signaling pathways initiating from both inside and outside the cell, culminating in the activation of caspases that execute the final stages of cell death. Various methods of apoptosis prevention have been used to maintain cell viability during extended production runs in mammalian cell culture (Arden and Betenbaugh, Trends Biotechnol. 22:174-180 (2004); Vives et al., Metab. Eng. 5:124-132 (2003)). Altering the extracellular environment through media supplementation of growth factors, hydrolysates, and limiting nutrients has led to increased protein production and decreased apoptosis (Burteau et al., In Vitro Cell Dev. Biol. Anim. 39:291-296 (2003); Zhang and Robinson, Cytotechnology 48: 59-74 (2005)). Other researchers have turned to chemical and genetic strategies to inhibit the apoptotic signaling cascade from within the cell (Sauerwald et al., Biotechnol. Bioeng. 77:704-716 (2002), Biotechnol. Bioeng. 81:329-340 (2003)). Researchers have found that over-expression of genes found upregulated in cancer cells can prolong viability in cells grown in bioreactors by preventing apoptosis upstream of caspase activation (Goswami et al., supra; Mastrangelo et al., Trends Biotechnol. 16:88-95 (1998); Meents et al., Biotechnol. Bioeng. 80:706-716 (2002); Tey et al., J Biotechnol. 79:147-159 (2000) and Biotechnol. Bioeng. 68:31-43 (2000)).
The anti-apoptotic genes that function in the mitochondrial apoptotic pathway can be divided into three groups, namely 1) those that act early in the pathway, e.g., members of the Bcl-2 family of proteins; 2) those that act mid-pathway to disrupt or inhibit the apoptosome complex, e.g., Aven and 3) those that act late in the pathway, e.g., caspase inhibitors, XIAP. The functionality of the majority of these genes have been studied by over-expressing them in mammalian expression systems, and in some cases the effect of combined over-expression of two or more genes, each derived from a different part of the pathway has been determined. Examples include 1) the additive effect of Bcl-XL and a deletion mutant of XIAP (XIAPΔ) in CHO cells (Figueroa et al., Metab. Eng. 5:230-245 (2003)); 2) E1B-19K and Aven in BHK cells (Nivitchanyong et al., Biotechnol. Bioeng. 98:825-841 (2007)) and 3) Bcl-XL, Aven and XIAPΔ (Sauerwald et al., Biotechnol. Bioeng. 81:329-340 (2003)).
One of the major activators of the apoptosis cascade is the protein p53. One mechanism by which p53 activates apoptosis is through up-regulation of a subset of pro-apoptosis proteins including BNIP3 (Yasuda et al., J. Biol. Chem. 273:12415-21 (1998)). Therefore, up-regulation of p53 may be one of the principal factors triggering apoptosis. p53 can be degraded in cells through ubiquitin mediated degradation pathway via MDM2 (murine double minute-2 gene) (Bond et al., Current Cancer Drug Target 5:3-8 (2005)). Thus, over-expression of MDM2 has the potential to lower p53 levels and by extension, inhibit apoptosis. Previously, it was shown that in the presence of stress signals, a CHO cell line over-expressing MDM2 could survive longer in culture compared to wild type CHO in batch culture (Arden et al., Biotechnol. Bioeng. 97:601-614 (2007)).
The various approaches described above to increase protein productivity have succeeded to varying degrees. Nevertheless, there is a continuous need to develop methods to increase protein production, especially in large-scale commercial production.